Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain

Buelt, Melissa K.; Bernlohr, David A.

doi:10.1021/bi00484a008

Cited by 29 publications

(25 citation statements)

References 24 publications

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“…To do this, stopped-flow kinetic analysis of the modification of Cys 117 by DTNB was carried out. Previous work from our laboratory had shown that the side chain of Cys 117 is located within the ligand binding cavity of ALBP (22), but nonetheless could be covalently modified by the thiol reactive agent 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB) (13). DTNB is itself large and bulky, and cysteine modification would be expected to be dependent on the accessibility of the reagent to Cys 117 .…”

Section: -Ray Diffraction Results At Two Ph Values From the Mutant Almentioning

confidence: 99%

“…All preparations of ALBP were found to be apoprotein as revealed by Cys 117 titration. For ALBP, fatty acid binding blocks Cys 117 modification and conversely, Cys 117 modification blocks fatty acid binding (13), lowering the pH to 5.0 releases bound fatty acids and allows the titration of the sulfhydryl group.…”

Section: Methodsmentioning

confidence: 99%

“…Denaturation Studies-Equilibrium unfolding as a function of increasing concentration of guanidine HCl was carried out as described previously (13). Briefly, V32D/F57H ALBP or wild-type ALBP were dialyzed into either 50 mM MES, pH 5.5, or 50 mM Tris-HCl, pH 8.0, and then diluted to 0.5 M in the appropriate buffer at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…Sulfhydryl Modification-Modification of Cys 117 was carried out as described previously (13). Briefly, protein was added to a final concentration of 10 M in 50 mM Tris-HCl, pH 8.5, 100 mM NaCl and reacted with 80 M DTNB at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Ory

Kane

Simpson

et al. 1997

Journal of Biological Chemistry

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A number of crystallographic studies of the adipocyte lipid-binding protein have established that the fatty acid-binding site is within an internalized water-filled cavity. The same studies have also suggested the existence of a region physically distinct from the fatty acid-binding site which connects the cavity of the protein with the external solvent, hereafter referred to as the portal. In an effort to examine the portal region, we have used site-directed mutagenesis to introduce the mutations V32D/F57H into the murine ALBP cDNA. Mutant protein has been isolated, crystallized, and its stability and binding properties studied by biochemical methods. As assessed by guanidine-HCl denaturation, the mutant form exhibited a slight overall destabilization relative to the wild-type protein under both acid and alkaline conditions. Accessibility to the cavity in both the mutant and wild-type proteins was observed by stopped-flow analysis of the modification of a cavity residue, Cys 117 , by the sulfhydryl reactive agent 5,5 -dithiobis(2-nitrobenzoic acid) at pH 8.5. Cys 117 of V32D/F57H ALBP was modified 7-fold faster than the wild-type protein. The ligand binding properties of both the V32D/F57H mutant and wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation constants for 1-anilinonaphthalene-8-sulfonic acid were approximately 9 -10-fold greater than the wild-type protein, independent of pH. In addition, there is a 6-fold increase in the K d for oleic acid for the portal mutant relative to the wild-type at pH 8.0. To study the effect of pH on the double mutant, it was crystallized and analyzed in two distinct space groups at pH 4.5 and 6.4. While in general the differences in the overall main chain conformations are negligible, changes were observed in the crystallographic structures near the site of the mutations. At both pH values, the mutant side chains are positioned somewhat differently than in wild-type protein. To ensure that the mutations had not altered ionic conditions near the binding site, the crystallographic coordinates were used to monitor the electrostatic potentials from the head group site to the positions near the portal region. The differences in the electrostatic potentials were small in all regions, and did not explain the differences in ligand affinity. We present these results within the context of fatty acid binding and suggest lipid association is more complex than that described within a single equilibrium event.

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Section: -Ray Diffraction Results At Two Ph Values From the Mutant Almentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Ory

Kane

Simpson

et al. 1997

Journal of Biological Chemistry

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“…Chemical modification has shown that the relative size of the residue at position 117 has a significant effect on fatty acid binding [18]. This…”

Section: Figure 5 Phylogenetic Relationships Fabp Sequencesmentioning

confidence: 91%

Two distinct types of fatty acid-binding protein are expressed in heart ventricle of Antarctic teleost fishes

Vayda

Londraville

Cashon

et al. 1998

Biochemical Journal

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This report provides the first evidence for the existence of two distinct types of fatty acid-binding protein (FABP) in cardiac tissue of vertebrates. Four species of Antarctic teleost fish (Chaenocephalus aceratus, Cryodraco antarcticus, Gobionotothen gibberifrons and Notothenia coriiceps) exhibited two FABP mRNAs of 1.0 kb and 0.8 kb, which we have termed Hh-FABP and Had-FABP (isolated from eart tissue, with similarity to mammalian eart-type FABP or mammalian ipose-type FABP respectively). These FABP types appear to be products of distinct genes. Both FABP transcripts were abundant in cardiac and aerobic pectoral muscle. However, relative abundance of the two types varied distinctly among other tissues such as kidney, brain, spleen and white muscle. Neither FABP type was expressed in liver or intestine. The coding regions of Hh-FABP and Had-FABP cDNAs from the same species are only ~ 60% identical with one another. However, homologues of each FABP species, which exhibit > 98% identity to their respective types, were isolated from three other Antarctic teleosts. Phylogenetic analysis of aligned amino-acid sequences places Hh-FABP with other vertebrate heart-type FABPs, and Had with adipose/cutaneous FABPs. Expression of two distinct FABPs in cardiac tissue of Antarctic teleosts may be related to their ability to both utilize fatty acid as the primary metabolic fuel and to store lipid intracellularly.

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Table 7.IV

Yang¹,

Wu²,

Böhm³

Landolt-Börnstein - Group VII Biophysics

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Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain

Cited by 29 publications

References 24 publications

Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Biochemical and Crystallographic Analyses of a Portal Mutant of the Adipocyte Lipid-binding Protein

Two distinct types of fatty acid-binding protein are expressed in heart ventricle of Antarctic teleost fishes

Table 7.IV

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